Changes of Intramolecular Distances in the Na+/K+ Atpase upon Ouabain Binding

Abstract

The Na+/K+ ATPase is an integral membrane protein formed mainly by two subunits; α and β. Recently, we showed that ouabain (a cardiotonic steroid used in patients with heart failure) binds at two mutually exclusive sites along the ion permeation pathway of the Na+/K+ ATPase, indicating a sequential ouabain binding mechanism. Here, we used a spectroscopic approach (LRET) to measure distances between a genetically encoded lanthanide binding tag (LBT that binds Tb+3 with high affinity) and a cysteine-reactive fluorescent compound (TMR). We created energy transfer pairs by encoding the LBT's in several of the external loops of the α subunit and cysteines substituted in different positions of the β subunit. These normally functional LBT-Na+/K+ ATPase constructs were expressed in Xenopus laevis oocytes that were voltage clamped with two microelectrodes to obtain simultaneously electrical and LRET recordings under physiological ionic conditions. By measuring donor-only decays and sensitized emission decays, we estimated the distance between donor and acceptor. Distances from several LBT-(Tb3+)-TMR pairs were estimated, one at time, in the absence and then in the presence of ouabain. Interestingly, in the presence of ouabain, the distances increased. Ours results suggests that the Na+/K+ ATPase might transit to a different conformation (not yet detected by crystallography) when ouabain binds to the ion permeation pathway. This new conformation could be a separation between the α and β subunits or a rearrangement of the transmembrane segments to accommodate ouabain deep into the permeation pathway. Further experiments are required to discern between these possibilities. Supported by U54GM087519 and GM030376.